Molecule involved in binding of sperm to egg surfaces and procedures for use of this molecule to enhance or decrease potential fertility

ABSTRACT

A purified polypeptide which provides for initial binding of sperm to oocyte investments and has an active amino acid sequence of SEQ ID NO:12 (Cys-Gln-Ser-Leu-Gln-Glu-Tyr-Leu-Ala-Glu-Gln-Asn-Gln-Arg-Gln-Leu-Glu-Ser-Asn -Lys-Ile-Pro-Glu-Val-Asp-Leu-Ala-Arg-Val-Val-Ala-Pro-Phe-Met-Ser-Asn-Ile-Pro-Leu-Leu-Leu-Tyr-Pro-Gln-Asp-Arg-Pro -Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn-Glu-Asp-Val-Cys); or SEQ ID NO:13 (Cys-Glu-Ser-Leu-Gln-Lys-His-Leu-Ala-Glu-Leu-Asn-His-Gln-Lys-Gln-Leu-Glu-Ser-Asn-Lys-Ile-Pro-Glu-Leu -Asp-Met-Thr-Glu-Val-Val-Ala-Pro-Phe-Met-Ala-Asn-Ile-Pro-Leu-Leu-Leu-Tyr-Pro-Gln-Asp-Gly-Pro-Arg-Ser-Lys-Pro-Gln -Pro-Lys-Asp-Asn-Gly-Asp-Val-Cys); or the shorter but biologically active SEQ ID NO:1 and SEQ ID NO:9 (Tyr-Pro-Gln-Asp-Arg-X-Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn, where X is Thr or Pro). The polypeptide is useful in improving sperm-egg binding by fresh sperm, restoring sperm-egg binding following a cryopreservation cycle, enhancing fertilizing potential, and producing antibodies to determine fertilizing potential of sperm, determining sperm-binding sites on egg investments, and contraception.

FIELD OF THE INVENTION

This invention relates to sperm-egg binding proteins and their uses.

BACKGROUND OF THE INVENTION

Assisted reproductive technologies such as artificial insemination havebeen practiced commercially with livestock and horses for almost acentury, and with poultry, humans and other species for almost a halfcentury. Artificial insemination requires a sample of semen consistingof spermatozoa produced by the testes and seminal plasma contributed byepithelial cells of the epididymides, deferent ducts and accessory sexglands. In addition to water, seminal plasma contains numerous proteinsand glycoproteins, phospholipids, lipids, sugars and othercarbohydrates, and ions.

It was early recognized that those portions of seminal plasmaoriginating from the deferent ducts and accessory sex glands are notessential for spermatozoa to acquire or to retain fertilizingcapability. Furthermore, for some species, or for certain males of aspecies, retention of trace or moderate amounts of seminal plasma inassociation with the sperm is desirable for retention of fertilizingcapability during storage, whereas for others seminal plasma isdeleterious. The beneficial or deleterious effects of seminal plasma onsperm became increasingly evident as procedures were developed to storesperm for several hours or days at 4 to 22° C., or for years at -196° C.

It has become evident that certain proteins or glycoproteins,phospholipids, and other components of seminal plasma can be looselybound to sperm, presumably by interaction with the glycocalyxsurrounding the sperm plasma membrane, or less frequently by trueincorporation into the plasma membrane. Such molecules might be involvedin binding of sperm to eggs. It has been reported that a crude mixtureof proteins, extracted from human sperm by treatment with 0.6M KCl,increased the number of human sperm bound to a human zona pellucida invitro. Jean et al., "Increased Zona-binding Ability After Incubation ofSpermatozoa with Proteins Extracted from Spermatozoa of Fertile Semen",J. of Reproduction and Fertility (1995), Vol. 105, pp. 43-48. However,in contrast to the present invention, that protein was not obtained byfreezing sperm, and that publication gives no clue to the identity ofthe active molecule(s), mechanism of action, or species specificity.

The initial event in the fertilization process is binding of one or moresperm to the egg investments. Most research has focused on the molecularnature of the egg coverings in mammals (zona pellucida, an acellularcoating outside a mammalian oocyte, and plasma membrane) and the natureof enzymes or glycoproteins on or in the plasma membrane or acrosome ofsperm in mammals and invertebrates. Little research has been done on themolecular nature of avian sperm-egg binding, especially as it relates tothe "sperm side" of the interaction.

In mammals, a spermatozoon is considered to bind to the oocyte through aseries of egg-binding proteins (ligands) located on the surface of thespermatozoon which interact with appropriate members of a series ofsperm receptors located on the investments of the oocyte, namely thezona pellucida and oocyte plasma membrane.

Sperm-egg binding proteins generally are considered to be transmembraneproteins or glycoproteins, possibly with enzymatic activity, with anextracellular domain that interacts with a specific sperm receptor. Theconsensus scenario which has evolved is one of sequential binding: (a)loose binding to the zona pellucida via one or more molecules located onthe sperm plasma membrane, (b) tight binding to the zona pellucida via(a) molecule(s) located on the plasma membrane and/or the acrosomalmembrane, and (c) tight binding to the oocyte plasma membrane followedby fusion of the sperm and egg plasma membranes and entrance of theentire spermatozoon into the oocyte. Thus, species-specific adhesionbetween sperm and eggs is attributable to complexes formed betweenegg-binding proteins on sperm and complementary sperm receptors on eggs.Species non-specific egg-binding proteins are thought to exist, becausebinding to zona-free hamster oocytes occurs with sperm of many species,but at least one species-specific egg-binding protein also must belocated on the sperm surface. To date, no universal class of candidatemolecules has been identified as the species-non-specific, looseegg-binding component.

SUMMARY OF THE INVENTION

The present invention is a purified polypeptide (termed universalprimary sperm-egg binding protein, or UPSEBP) which provides for initialbonding of sperm to oocyte investments and uses of the polypeptide. Theinventive polypeptide has biological activity in a variety of avian andmammalian species and has active sites within the amino acid sequenceembodied in SEQ ID NO:12 which isCys-Gln-Ser-Leu-Gln-Glu-Tyr-Leu-Ala-Glu-Gln-Asn-Gln-Arg-Gln-Leu-Glu-Ser-Asn-Lys-Ile-Pro-Glu-Val-Asp-Leu-Ala-Arg-Val-Val-Ala-Pro-Phe-Met-Ser-Asn-Ile-Pro-Leu-Leu-Leu-Tyr-Pro-Gln-Asp-Arg-Pro-Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn-Glu-Asp-Val-Cys.One of these active sites is within the portion of the amino acidsequence embodied in SEQ ID NO:9 orTyr-Pro-Gln-Asp-Arg-X-Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn, where X isThr or Pro. The tertiary structure of SEQ ID NO:12 or a similar sequenceaffects biological activity. Uses of the synthetic or naturalpolypeptide include in vitro treatment of sperm to restore fertilizingcapacity for some samples of thawed cryopreserved sperm or to enhancefertilizing potential of some fresh sperm, intra-vaginal treatment ofsperm to enhance sperm-egg binding and increase probability offertilization, and use in an in vitro assay to determine fertilizingpotential of sperm. Antibodies to the polypeptide are also useful for invitro assays to determine potential fertility of sperm or number ofsperm-binding sites on an egg investment or in vivo as a contraceptive.

There exists a need for a universal sperm-egg binding material for usein enhancing the ability of sperm to bind loosely with an egg or otherfertilization or contraception techniques.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 compares the number of sperm bound before and aftercryopreservation, treated with UPSEBP and untreated to fresh sperm;

FIG. 2 compares the restorative ability of SGP-1 (prosaposin) andsaposins A, B, C and D when used to treat frozen-thawed rooster sperm;

FIG. 3 compares the reduction in number of rooster sperm bound aftertreatment of sperm with polyclonal antibody against rat SGP-1, saposinsA, B, C and D, and a portion of the intervening sequence between saposinA and saposin B;

FIG. 4 compares the percentages of eggs which were fertilized (Trial 1)and hatched to provide a chick (Trial 2), laid by hens artificiallyinseminated with fresh sperm, frozen-thawed sperm and treatedfrozen-thawed rooster sperm;

FIG. 5a shows the fertility of 20 individual roosters, as the percentageof eggs laid providing a living chick, when 18 hens/male wereinseminated with fresh sperm and 18 other hens/male were inseminatedwith treated fresh sperm from the same ejaculate, with 3 replicateinseminations;

FIG. 5b shows the percentage increase in fertility resulting fromtreatment of sperm with UPSEBP for the 20 roosters of FIG. 5a;

FIG. 6 shows ranges of amounts of UPSEBP (crude extract of nativeprotein) which partially restored binding capability to frozen-thawedrooster sperm;

FIG. 7a shows the restorative capability of rooster UPSEBP (nativeprotein extracted from rooster sperm) and rat SGP-1 on binding of thawedcryopreserved turkey sperm;

FIG. 7b shows the restorative capability of turkey UPSEBP (nativeprotein extracted from turkey sperm) on binding of thawed cryopreservedrooster sperm and thawed cryopreserved turkey sperm;

FIG. 8 shows the capability of rooster UPSEBP and turkey UPSEBP torestore the binding of turkey sperm stored at 4° C. for 24 hours; and

FIG. 9 shows the restorative capability of rooster UPSEBP (nativeprotein extracted from rooster sperm) on binding of thawed cryopreservedstallion sperm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a native or synthetic polypeptide whichprovides for sperm-egg binding and the uses of the polypeptide. Thepolypeptide of the present invention is a member of the family ofproteins including prosaposin (also termed SGP-1) and saposins.Prosaposin has been reported in human seminal plasma at 90 μg/ml and haslong carbohydrate side chains. Four saposins (A, B, C, D) are producedby proteolytic cleavage of prosaposin, and individually have molecularweights of approximately 15 kilodaltons. The three residual interveningsegments between saposins A, B, C and D have no known function. Theparent prosaposin and processed saposins have been detected in almostevery tissue examined, including the brain, testes, heart, kidney,liver, spleen, semen and milk.

The native polypeptide of this invention was purified by centrifugationof sperm suspensions frozen and thawed in the presence of 10 to 12percent glycerol, which provided a supernatant fraction which was passedthrough a 300 kilodalton cut off ultrafiltration membrane. The filtratewas extracted with N-butanol: diisopropylether to remove lipid andprovide a crude protein preparation. Addition of this crude protein to asuspension of frozen-thawed rooster sperm restored their ability to bindto the membrane preparations from hen's eggs. The active moiety of thenative polypeptide was concentrated in the aqueous phase after lipidextraction and had an apparent molecular weight of about 10 kilodaltons.

The active molecule represented less than 0.1 percent of the crudeprotein recovered from the N-butanol: diisopropylether extracts, and wasincreased in specific activity by use of a G3000-SWXL gel filtrationcolumn followed by C4-hydrophobicity chromatography.

Hereafter, the molecule, originally isolated from rooster sperm, istermed "universal primary sperm-egg binding protein" or "UPSEBP." Theamino acid sequence SEQ ID NO:1(Tyr-Pro-Gln-Asp-Arg-Thr-Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn) was foundto confer substantial binding activity as did SEQ ID NO:9(Tyr-Pro-Gln-Asp-Arg-Pro-Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn) and SEQ IDNO:12 (Cys-Gln-Ser-Leu-Gln-Glu-Tyr-Leu-Ala-Glu-Gln-Asn-Gln-Arg-Gln-Leu-Glu-Ser-Asn-Lys-Ile-Pro-Glu-Val-Asp-Leu-Ala-Arg-Val-Val-Ala-Pro-Phe-Met-Ser-Asn-Ile-Pro-Leu-Leu-Leu-Tyr-Pro-Gln-Asp-Arg-Pro-Arg-Ser-Gln-Pro-Gln-Pro-Lys-Ala-Asn-Glu-Asp-Val-Cys). SEQID NO:12, which contains SEQ ID NO:9, conferred substantial bindingactivity especially when oxidized to form a molecule with a hairpinform. These amino acid sequences are compared in Table No. 1 withpertinent portions of the amino acid sequences of the similar rat SGP-1(SEQ ID NO:14). Human prosaposin (SEQ ID NO:15), and mouse prosaposin(SEQ ID NO:16) also are shown in Table 1. Neither SEQ ID NO:1 nor SEQ IDNO:9 overlaps with the amino acid sequences of saposin A or saposin B,and SEQ ID NO:12 is the entire intervening sequence saposin A-B plus theterminal 6 amino acids of saposin A and the first 4 amino acids ofsaposin B. The biological activity of SEQ ID NO:12 or UPSEBP is distinctfrom those ascribed to saposin A, saposin B, or other regions of theintact prosaposin molecule. SEQ ID NO:13 is a variation of SEQ ID NO:12and also should have high biological activity.

The biological activity of the native UPSEBP prepared from rooster spermand synthetic amino acid sequences were compared in an in vitro assay ofsperm binding described in copending U.S. patent application Ser. No.08/234,448. This microwell assay used plates in which the wells werecoated with a binding substrate prepared from an extract of hen's eggvitelline membrane. The thawed rooster sperm, known to be reduced inUPSEBP, were washed once with isotonic salt solutions, resuspended at50×10⁵ /0.1 ml, and incubated 30 minutes with 0.02-50 μg/0.5 ml nativeUPSEBP or synthetic peptide. Then 0.1 ml of a suspension of about 5×10⁶sperm were placed into each of triplicate wells in a microwell plate.After 2 hours of incubation at 37° C., unbound sperm were washed awayand bound sperm were stained and counted using a microscope. The numberof sperm bound was considered to be a measure of biological activitybecause there is a high correlation between the number of rooster spermbound in vitro to such an egg extract and the fertilizing potential ofsperm in that sample, as quantified by the percentage of eggs hatchingyoung chicks after artificial insemination.

Table No. 1 presents the amino acid sequence of synthetic peptides andthe resulting binding capacity as compared to native UPSEBP (fullsequence not provided). Initial evaluations of the capabilities of SEQID NO:1 through SEQ ID NO:11, native UPSEBP and rat SGP-1 to enhancesperm binding to an egg-membrane substrate were based on visual countsof bound sperm and conducted over a number of months. The binding datafor SEQ ID NO:1 through SEQ ID NO:12 and native UPSEBP and rat SGP-1presented in Table No. 1 were obtained as two data sets using a recentversion of that sperm-egg binding assay in which the bound sperm werestained with a fluorescent dye binding to DNA and quantified using afluorescent microwell-plate reader, by techniques known to those skilledin the art. The amino acid sequences of portions of human prosaposin(SEQ ID NO:15) and mouse prosaposin (SEQ ID NO:16) also are as shown.

A synthetic peptide having SEQ ID NO:1 provided sperm binding capabilitysimilar to that of native UPSEBP. Using nanomolar concentrations of SEQID NO:1, approximately twice as many sperm bound to the egg membranesubstrate. The slightly shorter amino acid sequence of SEQ ID NO:2 hadlower binding capability, and the very short SEQ ID NO:3 was essentiallydevoid of binding capability. The addition of amino acids distal to theterminal asparagine of SEQ ID NO:3 as embodied in SEQ ID NO:4 and SEQ IDNO:5 did not substantially change biological activity. Replacement ofthe terminal proline in SEQ ID NO:2 with a cysteine resulted in SEQ IDNO:6, with minimal change in biological activity relative to SEQ IDNO:2. Additions at the NH₂ end of an acetyl group in SEQ ID NO:7 orphenylalanine groups in SEQ ID NO:8 did not suppress biological activityrelative to SEQ ID NO:2. SEQ ID NO:1 consistently gave greaterbiological activity than SEQ ID NO:2 through SEQ ID NO:8.

Substitutions of threonine at amino acid number six in SEQ ID NO:1 byproline resulted in SEQ ID NO:9, which also had very high biologicalactivity, approximately equal to that of SEQ ID NO:1. The addition ofL-leucine to the NH₂ end of SEQ ID NO:1 resulted in SEQ ID NO:10 andaddition of D-leucine to the NH₂ end of SEQ ID NO:1 resulted in SEQ IDNO:11. The binding activity of SEQ ID NO:10 with the natural form ofleucine was greater than that of SEQ ID NO:11 although both of thesesequences had reduced activity relative to SEQ ID NO:1.

A synthetic peptide which embodied the complete intervening sequencebetween rat saposin A and saposin B was prepared as SEQ ID NO:12. SEQ IDNO:12 was studied in two forms. As synthesized, SEQ ID NO:12 had alinear, reduced form with SH-groups on the two terminal cysteine aminoacids; in this linear form SEQ ID NO:12 had substantial biologicalactivity and induced binding activity at 0.002 nM and, thus, had apotency approximately 90 times that of SEQ ID NO:9, which is entirelyembodied in SEQ ID NO:12, or approximately 85 times the potency of SEQID NO:1. Induced oxidation of the SH-groups in SEQ ID NO:12 modified itto a hairpin, or closed loop, form with an -S-S-linkage between the twoterminal cysteine amino acids. In this hairpin form SEQ ID NO:12 hadenhanced biological activity and induced binding activity at 0.0004 nMand, thus, had 5 times the activity of the linear form of SEQ ID NO:12and 425 times the activity of SEQ ID NO:1. Clearly, the hairpin form ofSEQ ID NO:12 was more active than the linear form and, thus, tertiarystructure as well as amino acid sequence are important for binding ofsperm to egg membranes by the molecule which is the subject of thisinvention.

The amino acid sequences of rat SGP-1 and human or mouse prosaposinspartially presented in Table 1 indicate that most of the SGP-1 orprosaposin molecule is not essential for sperm-egg binding activity.Furthermore, the portion of the prosaposin molecule with a sequencesimilar to that of SEQ ID NO:1 or SEQ ID NO:9 is an intervening sequenceproteolytically cleaved during processing of prosaposin, namely thedistal portion of the intervening sequence between saposin A and saposinB. Sequences integral to any of the saposins (A, B, C or D) are notnecessary for the biological activity of SEQ ID NO:1 or SEQ ID NO:9. SEQID NO:12 extends the intervening sequence between saposin A and saposinB for 6 amino acids in the NH₂ direction into saposin A and for 4 aminoacids in the COOH direction into saposin B. These extensions provide theterminal cysteines which are essential for formation of the hairpin loopform of SEQ ID NO:12. SEQ ID NO:5 combines the initial portion ofsaposin B with the terminal portion of the intervening sequence betweensaposin A and saposin B with virtually no biological activity.

Native or synthetic UPSEBP is useful for the following applications: a)as a pro-fertility additive to fluids used to suspend or resuspend spermat some point in the processing of semen for artificial insemination orfor in vitro fertilization or similar assisted reproductive technology;b) as the active ingredient for a vaginal pro-fertility medication forself administration; c) as the basis for analogs containing the epitopesproviding sperm binding but with sequences prohibiting successfulbinding to the egg investments for contraception; d) as the basis for areagent for use in quantifying the amount of UPSEBP present on sperm toprovide information related to the potential fertility of an individualspermatozoon or the population of sperm in a seminal sample; and e) asthe antigen for the production of antibodies useful in predictingpotential fertility.

In one embodiment, native or synthetic UPSEBP is used to improve thecapacity of fresh or frozen-thawed sperm to fertilize eggs. Prior toartificial insemination, an aliquot of fresh or frozen-thawed sperm ismixed with the native or synthetic UPSEBP which increases fertilizingcapacity of the sperm.

In another embodiment, native or synthetic polypeptide can be used as apro-fertility medication to be administered intra-vaginally as asemi-solid, liquid, or foam shortly before coitus. The medication mightcontain any of SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:13 or native UPSEBP as the active ingredient, suspended in anappropriate carrier which maintains biological activity, providesappropriate dispersion near the external cervical os, and facilitateseffective and rapid partition of the active ingredient to the spermafter ejaculation.

Analogs of the essential sequence within UPSEBP, such as SEQ ID NO:1,SEQ ID NO:9, SEQ ID NO:12, or SEQ ID NO:13, which contain the epitope(s)binding the molecule to sperm, and also contain alternative sequenceswhich prohibit rather than facilitate binding, are useful for theircontraceptive effect on sperm. A contraceptive medication containing theanalog is administered intra-vaginally as a semi-solid, liquid, or foamshortly before coitus. The medication containing a UPSEBP analog issuspended in an appropriate carrier which maintains biological activityand facilitates appropriate dispersion near the external cervical os andeffective and rapid partition of the active ingredient to the spermafter ejaculation.

In another embodiment, the amount of UPSEBP present on sperm prior toinsemination is quantified and is predictive of fertilizing potential.Synthetic sequences such as SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO:12, orSEQ ID NO:13 are used as the basis for a reagent used in quantifying theamount of UPSEBP present on sperm to provide information related topotential fertility of individual sperm or the population of sperm in aseminal sample. UPSEBP apparently binds to sperm in a stoichiometricmanner until saturation is reached. Exposure of sperm to the labeledmolecule enables quantification of unoccupied sperm sites for bindingUPSEBP and, indirectly, the number of UPSEBP molecules which had beenpresent on the sperm. The synthetic sequence (such as SEQ ID NO:1, SEQID NO:9, SEQ ID NO:12, or SEQ ID NO:13) of UPSEBP is conjugated withfluorescein isothiocyanate, Texas Red, or other cyanine dyes, each ofwhich fluoresces with a characteristic emission wavelength and intensitywhen excited with light of the proper wavelength. A suspension of spermis exposed to an excess of labeled SEQ ID NO:1, for example, forapproximately 5 minutes to enable binding, and then diluted >1:10,000and passed through a flow cytometer for quantification of the amount ofSEQ ID NO:8 bound by each of 3,000 to >10,000 sperm representative ofthose in the sample. A distribution profile can be made for thepercentages of sperm in the sample lacking or having enough UPSEBP priorto analysis, with the latter likely to have a high fertilizingpotential. Similar use could be made of a synthetic sequence or UPSEBPlabeled with a light-absorbing reporter.

In yet another embodiment, the invention encompasses antigens forproduction of antibodies useful in predicting potential fertility ofsperm. Natural or recombinant/synthetic UPSEBP or an analog thereof canbe used to produce monoclonal or polyclonal antibodies against one ormore epitopes of UPSEBP exposed while the molecule is bound by sperm.Antibodies against SEQ ID NO:6 and SEQ ID NO:12 have been produced andtested for use in such assays. Alternatively, a primary antibody againstSEQ ID NO:13, for example, can be cleaved into Fab-fragments, which thencan be directly labeled with a fluorescent cyanine dye before binding tothe sperm. Samples containing a high percentage of sperm which bind alarge amount of antibody have high fertilizing potential. Conversely,samples in which most sperm lacked typical amounts of UPSEBP would havepoor fertilizing potential. In this embodiment, appropriate antibodies(or fragments thereof) are labeled with a fluorescent or light-absorbingreporter group and then used as a reagent to quantify the number ofUPSEBP molecules present on each member of a population of sperm. Thesperm treated with labeled Fab-fragments of antibody can be placed intoan automated image analysis system in which both movementcharacteristics of individual sperm are quantified according totechnologies known to those skilled in the art, and concurrently thenumber of exposed binding sites for UPSEBP on each spermatozoon isdetermined on the basis of fluorescence as disclosed herein. Theresulting data establish a multidimensional distribution profile for thepercentages of sperm in the sample lacking or having enough UPSEBP priorto analysis to have a high fertilizing potential, and also the motioncharacteristics of each cell in terms such as velocity, head yaw orwobble, and linearity of motion. Such a multi-vector analysis of eachindividual member of the sample population is predictive of potentialfertility of that sample.

Although the invention has been described generally above, particularexamples give additional illustration of the products and method stepstypical of the present polypeptide and uses thereof. The data presentedin FIGS. 1 to 3 and 6 to 9 show the number of sperm bound per unit areaof laid hen's egg perivitelline membrane, or data derived from suchbinding, or bound to a substrate prepared using an extract from suchperivitelline membranes pursuant to the method disclosed in co-pendingpatent application Ser. No. 08/234,448. Except when noted, nativeprotein extracted from rooster sperm with UPSEBP activity was used.

EXAMPLE 1

Restoration of sperm binding capability

Freshly collected rooster sperm were cryopreserved, thawed and processedto remove glycerol. One portion of the thawed sperm received crudeUPSEBP at a concentration of 2 μg protein per 10⁶ sperm. Samplescontaining 1-10×10⁶ sperm were placed into wells coated with an extractof hen's egg membrane and incubated 180 minutes. Non-bound sperm wereremoved by washing, and bound sperm were stained withdiamido-2-phenylindole and counted using an epifluorescent microscope.The results presented in FIG. 1 demonstrate the ability of UPSEBP torestore the capability of frozen-thawed rooster sperm to bind to hen'segg membrane protein in comparison to unprocessed sperm.

EXAMPLE 2

Comparison of UPSEBP to SGP-1 and human saposins

The procedures of Example 1 were repeated using SGP-1 and human saposinsA, B, C and D. FIG. 2 shows that exposure of frozen-thawed sperm toSGP-1 restored binding to ≧100 percent of that obtained by exposure ofother aliquots of the same sample of frozen-thawed sperm to UPSEBP (2μg/million sperm) and that human saposin B restored binding to about 70percent of the level achieved using UPSEBP. Saposins A, C and D wereineffective at restoring binding.

EXAMPLE 3

Ability of polyclonal antibodies to neutralize sperm-egg binding

Polyclonal antibodies against rat SGP-1, human saposins and a portion ofthe intervening sequence between saposin A and saposin B were providedby others who had prepared the antibodies by techniques known in theart. Antibody and 12.5×10⁶ sperm were incubated for 15 minutes at roomtemperature. Sperm binding was determined as in Example 1. As shown inFIG. 3, antibodies against the intervening A-B sequence, saposin B andSGP-1 were very effective in eliminating activity of UPSEBP. Antibodyagainst saposin A was less effective, and antibodies against saposins Cand D had no effect.

EXAMPLE 4

Ability of UPSEBP to improve fertility of frozen thawed rooster sperm

Sperm in a sample of pooled semen were used to obtain fertility databefore cryopreservation (i.e., fresh) and after cryopreservation andpost-thaw processing to slowly remove glycerol (frozen-thawed). Oneportion of the thawed sperm was suspended in Minnesota-A buffer alone,and the second was treated with Minnesota-A buffer containing crudeUPSEBP at 2 μg protein per 10⁶ sperm. As shown in FIG. 4, fertility ofthe sperm processed in UPSEBP was double that for sperm not treated withUPSEBP and approached the level of fresh sperm. Hatchability of eggsartificially fertilized by sperm treated with UPSEBP increased from 18to 25 percent.

EXAMPLE 5

Ability of UPSEBP to improve fertility of fresh rooster sperm

Fresh sperm from 20 individual roosters were divided into two aliquots:one remained untreated, and the other was treated with native roosterUPSEBP at 0.04 μg protein per 10⁶ sperm. The percent fertility(percentage of ˜200 laid eggs which resulted in a live chick) andpercent increase in fertility over untreated sperm was determined and isdepicted in FIGS. 5a and 5b. For 8 out of the 20 roosters tested, theaddition of UPSEBP resulted in more than a 25 percent increase infertility, and for another 5 of the 20 roosters, there was a 7 to 23percent increase in fertility. The rooster sperm with lower naturallevels of fertility were most benefited by treatment with UPSEBP.

EXAMPLE 6

Amount of UPSEBP

Rooster sperm subjected to a cryopreservation cycle were treated withincreasing amounts of partially purified UPSEBP and subject to astandard binding assay, together with aliquots of unfrozen (fresh)sperm. FIG. 6 shows that treatment of frozen-thawed rooster sperm with0.04-4.0 μg UPSEBP per 10⁶ sperm partially restored the sperm'scapability to bind to the membrane of a hen's egg.

EXAMPLE 7

UPSEBP across poultry species

Frozen-thawed turkey sperm were exposed to rooster UPSEBP orsemi-purified SGP-1. Treatment of thawed frozen turkey sperm withrooster UPSEBP partially restored the capability of sperm to bind to amembrane of a hen's egg as depicted in FIG. 7a. Turkey UPSEBP, preparedin a manner similar to that used to prepare native rooster UPSEBP, alsopartially restored the capability of binding the turkey sperm or roostersperm to chicken egg membrane protein, as depicted in FIG. 7b.

EXAMPLE 8

Treatment of stored turkey sperm

Turkey sperm were stored for 24 hours at 4° C. Samples of the storedsperm were untreated or treated with 2 μg protein per 10⁶ sperm ofrooster UPSEBP or turkey UPSEBP. Samples of the treated and non-treatedsperm were tested at 2.5×10⁶ sperm/well for their capacity to bindchicken's egg membrane preparations together with samples of fresh orkilled sperm. As shown in FIG. 8, after 24 hours the untreated sperm haddepressed binding relative to the fresh sperm (0 hr.), and treatmentwith either rooster or turkey UPSEBP partially restored binding.

EXAMPLE 9

Treatment of stallion sperm

The ability of rooster UPSEBP to enhance binding of frozen-thawedstallion sperm to a chicken egg membrane preparation was evaluated.Sperm from two stallions of unknown fertility were frozen by aconventional procedure. One portion of each sample received nosupplement, while a second portion received 2 μg rooster UPSEBP per 10⁶sperm. FIG. 9 shows that for both seminal samples, rooster UPSEBP waseffective in increasing the percentage of stallion sperm bound to hen'segg membrane.

EXAMPLE 10

Antibody

A primary antibody was produced in two rabbits against SEQ ID NO:5,linked via the cysteine amino acid to keyhole limpet, with use ofstandard adjuvants and techniques. The immune serum was harvested andused as a primary antibody to label rooster sperm, fixed in low ionicstrength buffer using paraformaldehyde and processed by standardtechniques, after which fluorescein isothiocyanate-labeledgoat-antisheep gama globulin was used as a second antibody. Analyses byflow cytometry revealed the percent of sperm as labeled, and visualexaminations by epifluorescence microscopy revealed localization of theantibodies to the head region. A similar antibody was prepared againstSEQ ID NO:12.

Although the invention has been described with particularity in theabove text and examples, the invention is only to be limited insofar asis set forth in the accompanying claims.

                                      TABLE 1    __________________________________________________________________________    SEQUENCE COMPARISON OF SYNTHETIC PEPTIDES (SEQ ID NOS: 1 TO 13) WITH RAT    SGP-1    (SEQ ID NO: 14), HUMAN PROSAPOSIN (SEQ ID NO: 15) AND MOUSE PROSAPOSIN    (SEQ ID NO: 16)    AND DATA ON IN VITRO BINDING OF FROZEN-THAWED ROOSTER SPERM TREATED WITH    PEPTIDES    TO AN EXTRACT OF HEN'S EGG MEMBRANE BOUND TO WELLS OF A MICROWELL    PLATE.sup.\            SEQ ID NO.            1  2   3   4  5   6   7   8  9  10  11  12 13 14 15 16    __________________________________________________________________________    AMINO                                                 Pro                                                             Pro                                                                Pro    ACID                                                  Gly                                                             Gly                                                                Gly    SEQ.                                                  Glu                                                             Glu                                                                Glu                                                          Val                                                             Val                                                                Val                                                          Cys                                                             Cys                                                                Cys                                                          Ala                                                             Ala                                                                Ala                                                          Leu                                                             Leu                                                                Leu                                                          Asn                                                             Asn                                                                Asn                                                          Leu                                                             Leu                                                                Leu                                                    Cys                                                       Cys                                                          Cys                                                             Cys                                                                Cys                                                    Gln                                                       Glu                                                          Gln                                                             Glu                                                                Gln                                                    Ser                                                       Ser                                                          Ser                                                             Ser                                                                Ser                                                    Leu                                                       Leu                                                          Leu                                                             Leu                                                                Leu                                                    Gln                                                       Gln                                                          Gln                                                             Gln                                                                Gln                                                    Glu                                                       Lys                                                           Glu.sup.c                                                              Lys.sup.c                                                                 Glu.sup.c                                                    Tyr                                                       His                                                          .sup. Tyr.sup.d                                                             .sup. His.sup.d                                                                .sup.                                                                Tyr.sup.d                                                    Leu                                                       Leu                                                          Leu                                                             Leu                                                                Leu                                                    Ala                                                       Ala                                                          Ala                                                             Ala                                                                Ala                                                    Glu                                                       Glu                                                          Glu                                                             Glu                                                                Glu                                                    Gln                                                       Leu                                                          Gln                                                             Leu                                                                Gln                                                    Asn                                                       Asn                                                          Asn                                                             Asn                                                                Asn                                                       His   His                                                    Gln                                                       Gln                                                          Gln                                                             Gln                                                                Gln                                                    Arg                                                       Lys                                                          Arg                                                             Lys                                                                Lys                                                    Gln                                                       Gln                                                          Gln                                                             Gln                                                                Gln                                                    Leu                                                       Leu                                                          Leu                                                             Leu                                                                Leu                                                    Glu                                                       Glu                                                          Glu                                                             Glu                                                                Glu                                                    Ser                                                       Ser                                                          Ser                                                             Ser                                                                Ser                                                    Asn                                                       Asn                                                          Asn                                                             Asn                                                                Asn                                                    Lys                                                       Lys                                                          Lys                                                             Lys                                                                Lys                                                    Ile                                                       Ile                                                          Ile                                                             Ile                                                                Ile                                                    Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro                                                    Glu                                                       Glu                                                          Glu                                                             Glu                                                                Glu                                                    Val                                                       Leu                                                          Val                                                             Leu                                                                Val                                                    Asp                                                       Asp                                                          Asp                                                             Asp                                                                Asp                                                    Leu                                                       Met                                                          Leu                                                             Met                                                                Met                                                    Ala                                                       Thr                                                          Ala                                                             Thr                                                                Ala                                                    Arg                                                       Glu                                                          Arg                                                             Glu                                                                Arg                                                    Val                                                       Val                                                          Val                                                             Val                                                                Val                                                    Val                                                       Val                                                          Val                                                             Val                                                                Val                                                    Ala                                                       Ala                                                          Ala                                                             Ala                                                                Ala                                                    Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro                                                    Phe                                                       Phe                                                          Phe                                                             Phe                                                                Phe                                                    Met                                                       Met                                                          Met                                                             Met                                                                Met                                                    Ser                                                       Ala                                                          Ser                                                             Ala                                                                Ser                                                    Asn                                                       Asn                                                          Asn                                                             Asn                                                                Asn                                                    Ile                                                       Ile                                                          Ile                                                             Ile                                                                Ile                                                    Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro                                                    Leu                                                       Leu                                                          Leu                                                             Leu                                                                Leu                                      Phe           Leu                                                       Leu                                                          Leu                                                             Leu                                                                Leu                                      Phe   L-Leu                                                D-Leu                                                    Leu                                                       Leu                                                          Leu                                                             Leu                                                                Leu            Tyr               Tyr            Tyr Acetyl                                      Tyr                                         Tyr                                            Tyr Tyr Tyr                                                       Tyr                                                          Tyr                                                             Tyr                                                                Tyr                                  Tyr            Pro               Pro            Pro Pro Pro                                         Pro                                            Pro Pro Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro            Gln               Gln            Gln Gln Gln                                         Gln                                            Gln Gln Gln                                                       Gln                                                          Gln                                                             Gln                                                                Gln            Asp               Asp            Asp Asp Asp                                         Asp                                            Asp Asp Asp                                                       Asp                                                          Asp                                                             Asp                                                                Asp            Arg               Arg            Arg Arg Arg                                         Arg                                            Arg Arg Arg                                                       Gly                                                          Arg                                                             Gly                                                                His            Thr               Thr            Thr Thr Thr                                         Pro                                            Thr Thr Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro            Arg               Arg            Arg Arg Arg                                         Arg                                            Arg Arg Arg                                                       Arg                                                          Arg                                                             Arg                                                                Arg            Ser               Ser            Ser Ser Ser                                         Ser                                            Ser Ser Ser                                                       Ser                                                          Ser                                                             Ser                                                                Ser            Gln               Gln Gln Gln    Gln Gln Gln                                         Gln                                            Gln Gln Gln                                                       Lys                                                          Gln                                                             Lys                                                                Gln            Pro               Pro Pro Pro                          Pro Pro Pro Pro                                         Pro                                            Pro Pro Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro            Gln               Gln Gln Gln                          Gln Gln Gln Gln                                         Gln                                            Gln Gln Gln                                                       Gln                                                          Gln                                                             Gln                                                                Gln            Pro               Pro Pro Pro                          Pro Cys Pro Pro                                         Pro                                            Pro Pro Pro                                                       Pro                                                          Pro                                                             Pro                                                                Pro            Lys    Lys Lys                          Lys            Lys                                            Lys Lys Lys                                                       Lys                                                          Lys                                                             Lys                                                                Lys            Ala    Ala Ala                          Ala            Ala                                            Ala Ala Ala                                                       Asp                                                          Ala                                                             Asp                                                                Ala            Asn    Asn Asn                          Asn            Asn                                            Asn Asn Asn                                                       Asn                                                           Asn.sup.e                                                              Asn.sup.e                                                                 Asn.sup.e                       Glu                          Glu                       Glu                                                       Gly                                                          .sup. Glu.sup.                                                             .sup. Gly.sup.                                                                .sup.                                                                Glu.sup.                       Asp                          Asp                       Asp                                                       Asp                                                          Asp                                                             Asp                                                                Asp                       Val                          Val                       Val                                                       Val                                                          Val                                                             Val                                                                Val                       Cys                          Cys                       Cys                                                       Cys                                                          Cys                                                             Cys                                                                Cys                       Gln                          Gln                             Gln                                                             Gln                                                                Gln                       Asp                          Asp                             Asp                                                             Asp                                                                Asp                       Cys                          Cys                             Cys                                                             Cys                                                                Cys                       Met                          Met                             Met                                                             Ile                                                                Met                          Lys                             Lys                                                             Gln                                                                Lys                          Leu                             Leu                                                             Met                                                                Leu                          Val                             Val                                                             Val                                                                Val                          Thr                             Thr                                                             Thr                                                                Ser                          Asp                             Asp                                                             Asp                                                                Asp                                                          Ile                                                             Ile                                                                Val    PEPTIDE 0.17               300 2,125                       85 510 370 50  100                                         0.18                                            8.8 25  *     1.0                                                             Unk                                                                Unk    CONCEN-    TRATION.sup.a    ACTIVITY.sup.b            1  0.0006                   0.0001                       0.002                          0.0003                              0.0005                                  0.003                                      0.002                                         0.94                                            0.94                                                0.001     0.17                                                             Unk                                                                Unk    __________________________________________________________________________     .sup.a Concentration of peptide (nanomolar) which, after tenfold dilution     of the sperm suspension, provided maximum binding.     .sup.b Activity relative to SEQ ID NO: 1.     .sup.c End of saposin A.     .sup.d Start of intervening sequence between saposin A and saposin B.     .sup.e End of intervening sequence between saposin A and saposin B.     .sup.f Start of saposin B.     .sup.\ Maximum binding obtained with the native molecule in a     semipurified extract from rooster sperm gave a value of 80 sperm     (fluorescent) equivalent units/unit area; concentration of the active     molecule in the extract is unknown, but assuming MW = 10,000 and purity o     0.1% the mass used would have made a 1 nM solution. Sperm treated with SE     ID NO: 1 gave a value of 76 sperm (fluorescent) equivalent units/unit are     as compared to a value of 40 for untreated control sperm, and  #values fo     other sequences ranged from -40 to 141 sperm (fluorescent) units/unit are     at the designated concentration of peptide. Amino acids in rat SGP1     (prosaposin; Collard et al., 1988) are compared with those in human     prosaposin (O'Brien & Kishimoto, 1991; Kishimoto et al., 1992) and mouse     prosaposin (Tsuda et al., 1992).     *SEQ ID NO: 12 was studied in two forms: (1) as synthesized, in a linear     reduced form with SHgroups in the two terminal cysteines, and (2)     modified, to a hairpin oxidized form with an S-S-linkage between the two     terminal cysteines, induced by oxidation of the SHgroups. As a linear,     reduced, 60 amino acid molecule, SEQ ID NO: 12 was active at 0.002 nM and     thus, had ˜85 times the potency of  #SEQ ID NO: 1. As a hairpin,     oxidized, 60 amino acid molecule, however, SEQ ID NO: 12 was active at     0.0004 nM and, thus, had 425 times the potency of SEQ ID NO: 1. Thus, the     biological activity of SEQ ID NO: 12 was dependent on its tertiary     structure, with bioactivity activity of the hairpin loop form ˜5     times that of the linear form.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  16    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  15              (B) TYPE:  AMINO ACI - #D              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Tyr Pro Gln Asp Arg Thr Arg Ser - # Gln Pro Gln Pro Lys    #   10    -      Ala Asn             15    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 12              (B) TYPE:  AMINO ACI - #D              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Tyr Pro Gln Asp Arg Thr Arg Ser - # Gln Pro Gln Pro    #   10    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH:  7              (B) TYPE:  AMINO ACI - #D              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Gln Pro Gln Pro Lys Ala Asn    #  5 1    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 15              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Gln Pro Gln Pro Lys Ala Asn Glu - # Asp Val Cys Gln Asp    #   10    -      Cys Met             15    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 19              (B) TYPE: AMINO ACID              (C) STRANDEDNESS: SINGLE              (D) TOPOLOGY: UNKNOWN    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Pro Gln Pro Lys Ala Asn Glu Asp - # Val Cys Gln Asp Cys    #   10    -      Met Lys Leu Val Thr Asp             15    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 12              (B) TYPE: AMINO ACID              (C) STRANDEDNESS: SINGLE              (D) TOPOLOGY: UNKNOWN    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Tyr Pro Gln Asp Arg Thr Arg Ser - # Gln Pro Gln Cys    #   10    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 12              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY: UNKNOWN    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Xaa Pro Gln Asp Arg Thr Arg Ser - # Gln Pro Gln Pro    #   10    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 14              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 8:    -      Phe Phe Tyr Pro Gln Asp Arg Thr - # Arg Ser Gln Pro Gln    #   10    -      Pro    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 15              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 9:    -      Tyr Pro Gln Asp Arg Pro Arg Ser - # Gln Pro Gln Pro Lys    #   10    -      Ala Asn             15    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 16              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 10:    -      Leu Tyr Pro Gln Asp Arg Thr Arg - # Ser Gln Pro Gln Pro    #   10    -      Lys Ala Asn            15    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 16              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 11:    -      Xaa Tyr Pro Gln Asp Arg Thr Arg - # Ser Gln Pro Gln Pro    #   10    -      Lys Ala Asn             15    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 60              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 12:    -      Cys Gln Ser Leu Gln Glu Tyr Leu - # Ala Glu Gln Asn Gln    #   10    -      Arg Gln Leu Glu Ser Asn Lys Ile - # Pro Glu Val Asp Leu    #         25    -      Ala Arg Val Val Ala Pro Phe Met - # Ser Asn Ile Pro Leu    #                 35    -      Leu Leu Tyr Pro Gln Asp Arg Pro - # Arg Ser Gln Pro Gln    #     50    -      Pro Lys Ala Asn Glu Asp Val Cys    #             60    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 61              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 13:    -      Cys Glu Ser Leu Gln Lys His Leu - # Ala Glu Leu Asn His    #   10    -      Gln Lys Gln Leu Glu Ser Asn Lys - # Ile Pro Glu Leu Asp    #         25    -      Met Thr Glu Val Val Ala Pro Phe - # Met Ala Asn Ile Pro    #                 35    -      Leu Leu Leu Tyr Pro Gln Asp Gly - # Pro Arg Ser Lys Pro    #     50    -      Gln Pro Lys Asp Asn Gly Asp Val - # Cys    #             60    - (2) INFORMATION FOR SEQ ID NO: 14:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 79              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 14:    -      Pro Gly Glu Val Cys Ala Leu Asn - # Leu Cys Gln Ser Leu    #   10    -      Gln Glu Tyr Leu Ala Glu Gln Asn - # Gln Arg Gln Leu Glu    #         25    -      Ser Asn Lys Ile Pro Glu Val Asp - # Leu Ala Arg Val Val    #                 35    -      Ala Pro Phe Met Ser Asn Ile Pro - # Leu Leu Leu Tyr Pro    #     50    -      Gln Asp Arg Pro Arg Ser Gln Pro - # Gln Pro Lys Ala Asn    #             65    -      Glu Asp Val Cys Gln Asp Cys Met - # Lys Leu Val Thr Asp    #   75    -      Ile    - (2) INFORMATION FOR SEQ ID NO: 15:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 80              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 15:    -      Pro Gly Glu Val Cys Ala Leu Asn - # Leu Cys Glu Ser Leu    #   10    -      Gln Lys His Leu Ala Glu Leu Asn - # His Gln Lys Gln Leu    #         25    -      Glu Ser Asn Lys Ile Pro Glu Leu - # Asp Met Thr Glu Val    #                 35    -      Val Ala Pro Phe Met Ala Asn Ile - # Pro Leu Leu Leu Tyr    #     50    -      Pro Gln Asp Gly Pro Arg Ser Lys - # Pro Gln Pro Lys Asp    #             65    -      Asn Gly Asp Val Cys Gln Asp Cys - # Ile Gln Met Val Thr    #   75    -      Asp Ile             80    - (2) INFORMATION FOR SEQ ID NO: 16:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 79              (B) TYPE: AMINO ACID              (C) STRANDEDNESS:  SING - #LE              (D) TOPOLOGY:  UNKNOWN    -     (xi) SEQUENCE DESCRIPTION:  SEQ ID NO: - # 16:    -      Pro Gly Glu Val Cys Ala Leu Asn - # Leu Cys Gln Ser Leu    #   10    -      Gln Glu Tyr Leu Ala Glu Gln Asn - # Gln Lys Gln Leu Glu    #         25    -      Ser Asn Lys Ile Pro Glu Val Asp - # Met Ala Arg Val Val    #                 35    -      Ala Pro Phe Met Ser Asn Ile Pro - # Leu Leu Leu Tyr Pro    #     50    -      Gln Asp His Pro Arg Ser Gln Pro - # Gln Pro Lys Ala Asn    #             65    -      Glu Asp Val Cys Gln Asp Cys Met - # Lys Leu Val Ser Asp    #   75    -      Val    __________________________________________________________________________

What is claimed is:
 1. A purified synthetic polypeptide consisting ofSEQ ID NO:12 and SEQ ID NO:13.